Field of the Invention
This invention relates to assays to evaluate and/or determine the inactivation of biological material in/on a sample by quantifying the level of integrity, or alternatively, the degradation of, DNA and the materials necessary to perform the assays. The sample can be a food product (e.g., fruits, vegetables, meat from animals, or eggs) while the item can be any object (e.g., medical equipment, especially reusable medical equipment) for which one needs to determine that the amount of inactivation of specific hazardous biological material on the object or in a sample is at or below a pre-determined amount. Non-limiting examples of hazardous biological material are toxins, viruses, parasites, fungi, bacteria, spores (bacterial, fungal or parasitical) and cancer cells. This invention further relates to the tools used in the assays. One assay uses quantitative PCR and polynucleotide primers to assay the fragmentation of mitochondrial DNA found intrinsically in the food matrix. Another assay examines the size and fragmentation of total DNA present in the food matrix using any device which measures DNA fragment size globally. A third assay involves use of an extrinsic source of mitochondrial DNA added to the processing run in a recoverable container.
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “Perez-Diaz_seq_ST25_8-25-14.txt”, created on Oct. 25, 2014, and having a size of 6 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
Description of the Prior Art
In order to render food products (both processed and fresh food products) safe and to prevent them from spoiling under the usual conditions of storage, some form of commercial sterilization is required. Control of food-borne bacterial pathogens such as Salmonella spp., Shigella spp., pathogenic Escherichia coli, Campylobacter spp., and Yersinia spp. has traditionally been achieved using heat treatments such as pasteurization or pressure sterilization. Heat treatments higher than pasteurization are used to provide Clostridium botulinum safety in low-acid canned foods and canned cured meats. Low-acid products have a pH of approximately 4.6 and above and include most meat and marine products, corn, peas, lima beans, asparagus and spinach. More recently, inhibition of surviving Clostridium botulinum spores has been achieved by treating food with NaNO2 and/or potassium sorbate. Failure to adequately inactivate Clostridium botulinum spores can result in the production of neurotoxins by the bacteria. Of course, failure to destroy other disease causing bacteria (e.g., Salmonella spp., Shigella spp., pathogenic E. coli, Campylobacter spp., and Yersinia spp.) in food products can result in food poisoning and severe disease after ingesting the contaminated food products. In addition, butchered meats and raw eggs can become contaminated with pathogenic bacteria during the handling and/or processing of the meats and eggs. It is vital to reduce or eliminate the bacteria on the surface of these items so that the workers and people that consume the food are not sickened by the bacteria.
Food spoilage and food poisoning from contamination of food by bacteria is a major problem throughout the world, including developed countries such as the United States, Canada, Japan, U.K., France, Germany, and Russia. In the U.S. alone, illness from food-borne bacteria costs several billion dollars annually in morbidity and mortality. Gram-positive and Gram-negative food-borne bacteria account for many of the pathogens causing food poisoning.
Traditional safety guidelines have traditionally used destruction of bacterial surrogates as indicators of processing safety and efficacy. Culturing samples of food or items for viability of bacteria and/or spores is a current method for assaying the destruction of bacterial surrogates. Two bacterial surrogates are Geobacillus stearothermophilus and Bacillus subtilis spores which are placed in or on samples prior to inactivation and which are cultured after treatment to determine if the bacteria and/or the bacterial spores have been inactivated. Problems with the culture approach include tracking and recovering indicator spores and/or bacteria, excessive time required to culture (48 hours or more), and molecular methods unable to differentiate between live and dead surrogates. Other devices and methods have been created to assay for live or killed bacteria and bacterial spores in food and devices using various culturing conditions (e.g., US Pub. 2007-0249040; U.S. Pat. Nos. 5,486,459; 5,830,683; 5,989,852; and WO 1995/008639), yet these methods and devices lack the ability to timely and accurately determine the amount of reduction of viable hazardous biological materials in or on a food or item.
To overcome the limitation of the long incubation times required for the proliferation of microbes and/or bacterial spores, time and temperature integrator (TTI) technologies have been developed. An example of such technologies is the application of enzymes derived from microbes that can naturally tolerate high temperatures and need such high temperatures to proliferate. For example, α-amylase from Bacillus licheniformis (Cordt, et al., J. Chem. Tech. and Biotech. 59:193-199 (1994)), lipase (U.S. Pat. No. 4,284,719), and β-glucosidase (Adams and Langley, Food Chemistry, 62:65-68 (1998)) have been used as TTIs. These enzymes can be produced in high concentrations using molecular biology techniques. The pure enzyme is then encapsulated in plastic tubing with 1 mm diameter, and the ends are sealed by melting. The encapsulated enzyme can be incorporated in a food matrix and recovered after thermal treatment (e.g., pasteurization) of the food matrix is applied. The activity of the post-thermal treated, heat resistant enzyme is determined and correlated with the effectiveness of the heating step. This rapid method allows for evaluation of thermal treatments uniformity and effectiveness. However, production of enzymatic TTI is complex. Also, TTI's enzymatic activity is difficult to maintain during long storage periods and may vary within multiple production batches.
Alternative approaches to culturing bacteria and enzymatic TTI, include assays for bacterial DNA or mRNA in samples of food using PCR and PCR-related techniques. See, e.g., U.S. Patent App. Pub. 2010-0167956 in which polynucleotide probes specific for E. coli are placed on a chip for assaying for the presence of E. coli; and U.S. Patent App. Pub. 2012-0288864 in which S. enterica, a food-borne pathogen, is detected by PCR using primers specific for an S. enterica gene.
The effect of high temperature on DNA degradation is well described. Above 100° C. denaturation, depurination, deamination and loss of secondary structure occurs (Gryson, N., Anal. Bioanal. Chem. 396:2003-2022 (2010)). Although autoclaving a foodstuff at 121° C. for fifteen minutes does not destroy all DNA available for PCR (Lipp, et al., J. AOAC Int. 82(4):923-928 (1999)), recovery of reduced DNA concentrations via quantitative PCR (qPCR) from cornmeal and water cooked for sixty minutes at 100° C. have been reported (Murray, et al., J. of Agric. & Food Chem. 55:2231-2239 (2007)). Increased Ct (threshold cycles) values occurred in DNA from heat-treated corn grits and corn flour when compared to untreated corn and resulted in distortions of qPCR assays for detection of genetically modified organisms (GMO) (Moreano, et al., J. of Agric. & Food Chem. 53:9971-9979 (2005)).
However, these prior art methods for detecting bacteria via PCR are unable to determine if the bacteria or their spores are still viable or not because PCR often is able to detect a certain fragment of DNA from the bacteria or spores, despite their death or inactivation. In a study by Stam (Doctoral Thesis, NCSU Food Science; Raleigh, N.C. (2008), available at http://www.lib.ncsu.edu/resolver/1840.16/3192), Clostridium sporogenes spores were heat-treated to 121° C. in two minutes intervals for eighteen minutes, and bacterial DNA degradation over time was determined. It was noted that heat-treating the spores for only two minutes resulted in the absence of DNA bands using agarose gel electrophoresis. However, the autoclaved spore DNA was still detectable by qPCR, having a reduced Ct value of 35 compared to a Ct of 12 for viable spores (Stam (2008)). Therefore, bacterial or spore DNA is degraded but still detectable by qPCR, when using thermal processing techniques such as heat or microwave suitable for preserving vegetables and fruits. Obviously, the repercussions of being wrong about viability of biological material in or on food or other items are serious and could result in serious morbidity and possible mortality in humans. It can also lead to false positives, which have a negative financial impact on the food industry. Also, excessively heating food matrices to destroy bacterial or bacterial spores DNA is also problematic because the quality of the texture, flavor and appearance of the finish good is reduced.
In addition to food substances, many other items need to be rendered sterile or have a reduction in the viability of biological material on the items prior to usage. For example, reusable medical and dental devices (such as, but not limited to, endoscopes, catheters, sponges, clamps, scalpels, drills, and suction tubes) need to be cleaned (biological material inactivated) after being used on one subject, prior to use on another subject. Biological material present on robust medical equipment is often inactivated by subjecting the contaminated equipment to high temperatures and pressure via a steam autoclave. While such inactivation methods are very effective for more durable medical instruments, advanced medical instruments formed of rubber and plastic components with adhesives are delicate and wholly unsuited to the high temperatures and pressures associated with a conventional steam autoclave. Thus, some steam autoclaves have been modified to operate under low pressure cycling programs to increase the rate of steam penetration into the medical devices or associated packages of medical devices undergoing cleaning. However, highly complex instruments which are often formed and assembled with very precise dimensions, close assembly tolerances, and sensitive optical components, such as endoscopes, may be destroyed or have their useful lives severely curtailed by harsh inactivation methods employing high temperatures and high or low pressures. Endoscopes, in particular, present problems in that such devices typically have numerous exterior crevices and interior lumens which can harbor microbes and thus be difficult to clean using ordinary techniques. The employment of a fast-acting yet gentle inactivation method is desirable for reprocessing sensitive instruments. Other medical or dental instruments which comprise lumens are also in need of methods of cleaning which employ an effective reprocessing system which will not harm sensitive components and materials. Further, the need exists for a reprocessing system having a shorter reprocessing cycle time. Regardless of how these devices are cleaned, failure to inactivate a substantial proportion of the biological material that may be present on the item after usage could result in the dangerous illness in the next subject on which the item is used.
The Food Safety Modernization Act (FSMA) mandates that companies document risk-based preventive controls for all pre-requisite programs as part of their Food Safety program. The Food and Drug Administration proposed guidelines under FSMA include the application of prevention standards to sanitation and environmental controls and monitoring. For example, one of the modifications under consideration involves sterilization of food packaging containers prior to filling the containers. Glass jars used for packaging of finished acidified and acid foods for the retail market are currently rinse with hot water, filled with the product, and subjected to a validated pasteurization step, often considered as a critical control point to render the finished food safe for consumption. New guidelines would require such containers to be subjected to a sterilization treatment prior to filling. The sterilization step for glass containers could be applied as a pasteurization, ultraviolet light, high pressure, or radiation treatment. The effectiveness of such treatments to eradicate pathogens of public health significance would have to be demonstrated after the treatments are applied.
As such, there remains a need for one or more assays that can evaluate and/or determine the amount of inactivation of biological material on/in food and/or an object in a timely and accurate manner. There is also a need for assays that can evaluate inactivation protocols and for evaluating deviations in processing to reduce the amount of viable biological material in/on items. The assays described herein use quantitative PCR. PCR and real-time PCR are well-known laboratory techniques and are accepted by AOAC International for clinical detection assays, including assays to detect BRCA1 and BRCA2 mutations.
Mitochondrial DNA (mtDNA) is used as identifiers in many scientific disciplines. They have been adopted for barcoding almost all groups of higher animals (http://www.barcoding.si.edu/). MtDNA is also used in human typing for forensic analysis (Hopwood, et al., Int. J. Legal Med. 108(5):237-243 (1996); Andreasson, et al., Biotechniques 33(2):402-411 (2002); Budowle, et al., Annu. Rev. Genomics Hum. Genet. 4:119-141 (2003)) using tissues such as bones, teeth, and hair shafts for DNA extraction. MtDNA primers or probes have been developed for source tracking fecal contaminates in wastewater influents and effluents using multiplex qPCR (Caldwell, et al., Environ. Sci. & Technol., 41:3277-83 (2007); Caldwell and Levine, J. Microbiol. Methods 77:17-22 (2009); Caldwell, et al., “Mitochondrial DNA as source tracking markers of fecal contamination”, In Microbial Source Tracking: Methods, Applications, and Case Studies, eds. Harwood, Hagedorn and Blanch (Springer Science and Business Media, NY) 229-250 (2011)). In the food industry, PCR-based mtDNA analyses are used in the authentication of food, and to trace contamination of other animals in the food products (Meyer and Candrian, Lebensm.—Wiss. u.—Technol. 29:1-9 (1996); Lahiff, et al., Mol. Cell Probes 15(1):27-35 (2001); Zhang, et al., Food Control 18:1149-1158 (2007); Fujimura, et al., Biosci. Biotechnol. Biochem. 72:909-913 (2008)). The development of those molecular tools improved the monitoring of food quality by preventing fraudulent description of food content, and identifying adulterants.